Many proteins of the extracellular matrix (ECM) are modified post-translationally by addition of oligosaccharide chains and are thus known as glycoproteins. The oligosaccharides are linked either O-glycosidically to serine or threonine residues, or N-glycosidically to an asparagine residue. Proteoglycans are glycoproteins that are substituted with a particular class of carbohydrate polymers, known as the glycosaminoglycans (GAGs). Proteoglycans are found in the ECM, at the cell surface and intracellularly in storage granules. In the ECM they contribute to the structure and organisation, and at the cell surface often function as receptors and/or co-receptors. All glycosaminoglycans (with the exception of hyaluronan) are synthesised on a core protein acceptor, and they are thus an integral component of proteoglycans (Wight et al., 1981; Heinegård and Paulsson, 1984, review).
Glycosaminoglycans (GAGs) are named to indicate that one of the monosaccharides in the repeating sequence of disaccharides is an amino sugar. The other monosaccharide is an uronic acid (glucuronic acid or iduroic acid), with the exception of keratan sulphate where it is a galactose. While other oligosaccharide substituents may be branched, GAG chains are linear (again, with the exception of keratan sulphate). Proteoglycans may be substituted with one (e.g. decorin) and up to some one hundred (e.g. aggrecan) GAG chains.
There are 4 types of glycosaminoglycans: hyaluronic acid, chondroitin sulphate/dennatan sulphate, heparan sulphate/heparin and keratan sulphate. The disaccharides in all glycosaminoglycan chains except hyaluronan are sulphated, increasing their negative charge and leading to an extended conformation of the chain. The molecule will occupy large solvent domains, observed as a high viscosity of a solution. This property is essential in cartilage and is the basis on which the tissue's resistance lies.
The repeating disaccharide sequence in CS is glucuronic acid-N-acetyl-galactosamine (GlcA-GalNAc), see FIG. 1. Chondroitin sulphate is found in several forms, named chondroitin-4 sulphate, -6 sulphate and -D and -E respectively. These forms differ in the sulphation of saccharides. CS-E is a highly sulphated species, which is attached to perlecan in the I and V domains.
FIG. 1. Basic structure of Chondroitin sulfate. Repeating dimeric ullits of GlcA β1-3 GalNAc. All hydroxy positions may be sulfated or/and epimerised.
The various positions open for sulfatation are numbered.
Chondroitin sulphate/dermatan sulphate is found in all extracellular matrices. Cartilage and invertebral disc are the tissues richest in chondroitin sulphate (Wight et al., 1981, review). Chondroitin sulphate is synthesised by specific enzymes located in the Golgi. The polymers are assembled onto a linker tri-saccharide. The hydroxyl group of serine residues followed by a glycine in the protein is substituted with a xylose and two successive galactose residues. Thereafter alternating monosaccharides of glucuronic acid and N-acetylgalactosamine are added successively to form the chain. Some glucuronate residues are converted to iduronate by an epimerase and sulfation is the last event just prior to secretion (Wight et al., 1981, review). In cartilage aggrecan, a member of the family hyalectins, is a chondroitin sulphate proteoglycan and is substituted with some one hundred CS chain and some thirty keratan sulphate chains. Aggrecan molecules are clustered along HA strands bound via their N-terminal globular domain. A protein known as link protein contacts both the HA-binding G1 domain of the aggrecan molecule and HA, and stabilises the complex. In this manner hundreds of aggrecan molecules are joined at one end to the HA. Thus, in cartilage matrix chondroitin sulphate is by far the most abundant GAG.
Perlecan was first identified as a large heparan sulphate proteoglycan isolated from the Engelbrecht-Holm-Swarm (EHS) murine basement membrane tumour. In basement membranes, it has been shown to bind several different classes of molecules. In each instance the core protein, the heparan sulphate (HS) side chains or both in concert, are involved in mediating the interaction. The proteoglycan binds to extracellular matrix components integral to basement membrane such as collagen IV, nidogen, laminin, and fibronectin (Timpl, R. and Brown, J. C. (1996) Bioassays 18, 123-132). Perlecan has also been shown to bind extracellular matrix components outside the basement membrane, e.g. PRELP and collagen type I (Bengtsson, E., Mörgelin, M., Sasaki, T., Timpl, R., Heinegård, D., and Aspberg, A. (2002) J. Biol. Chem). Perlecan supports cell-attachment both by binding and clustering integrins (Brown, J. C., Sasaki, T., Gohring, W., Yamada, Y., and Timpl, R. (1997) Eur. J Biochem. 250, 39-46). Binding to growth factors has been shown for both the HS side-chains (FGF-2 (Aviezer, D., Hecht, D., Safran, M., Eisinger, M., David, G., and Yayon, A. (1994) Cell 79, 1005-1013)) and the core protein (progranulin, (Gonzalez, E. M., Mongiat, M., Slater, S. J., Baffa, R., and Jozzo, R. V. (2003) J Biol Chem)). Based on its interactions, perlecan is assumed to have a role in basement membrane integrity.
Perlecan was originally thought to be substituted with HS exclusively, but later studies revealed that it is also present in a variant partially substituted with chondroitin sulphate (CS) (Couchman, J. R., Kapoor, R., Sthanam, M., and Wu, R. R. (1996) J Biol Chem 271, 9595-9602). Both the HS- and the HS/CS-substituted variants of perlecan have been found in tissues other than basement membrane, for example cartilage.
The generation of perlecan null mice revealed two particularly intriguing findings (Arikawa-Hirasawa, E., Watanabe, H., Takani, H., Hassell, J. R., and Yamada, Y. (1999) Nat Genet. 23, 354-358; Costell, M., Gustafsson, E., Aszódi, A., Morgelin, M., Bloch, W., Hunziker, E., Addicks, K., Timpl, R., and Fässler, R. (1999) J Cell Biol 147, 1109-1122). First, though mice lacking perlecan did develop grave disorders caused by compromised basement membrane strength or integrity (e.g. rupture of pericardial sac), the initial assembly of basement membranes seemed to be without complication. The second striking finding was the severe skeletal defects exhibited, apparently caused by the lack of perlecan in cartilage.
Following the publication of these results at least two human hereditary diseases with skeletal deficiencies have been ascribed to an underlying scarcity or complete lack of perlecan, underscoring the relevance of this finding in the mouse model (Nicole, S., Davoine, C. S., Topaloglu, H., Cattolico, L., Barral, D., Beighton, P., Hamida, C. B., Hammouda, H., Cruaud, C., White, P. S., Samson, D., Urtizberea, J. A., Lehmann-Horn, F., Weissenbach, J., Hentati, F., and Fontaine, B. (2000) Nat Genet. 26, 480-483; Arikawa-Hirasawa, E., Wilcox, W. R., Le, A. H., Silverman, N., Govindraj, P., Hassell, J. R., and Yamada, Y. (2001) Nat Genet. 27, 431-434).
In skeletal development, the deposition of a cartilaginous template precedes the formation of bones. The integrity of this template is a prerequisite for proper assembly of the skeleton. Perlecan-null mouse cartilage shows fewer and less organised collagen type II fibrils, and decreased levels of aggrecan, indicating a failure to organise the extracellular matrix (Costell, M., Gustafsson, E., Aszódi, A., Morgelin, M., Bloch, W., Hunziker, E., Addicks, K., Timpl, R., and Fäqssler, R. (1999) J Cell Biol 147, 1109-1122).
Mature collagen fibres may contain several different types of bound accessory proteins. They are part in the organisation of these fibres and regulate links to other molecules thereby contributing to the architecture of the fibrillar collagen network. A recent concept is that of modulator molecules, which regulate the early steps in the assembly of collagen monomers to fibres. Our laboratory has found that cartilage oligomeric matrix protein (COMP) accelerates the formation of fibres from monomers (Mörgelin and Heinegård, manuscript). Other molecules have the opposite effect and slow down fibre formation in vitro, e.g. decorin (Vogel, K. G., Paulsson, M., and Heinegård, D. (1984) Biochem. J. 223, 587-597) and fibromodulin (Hedborn, E. and Heinegård, D. (1989) J. Biol. Chem. 264, 6898-6905). Gene targeting of these molecules lead to abnormal collagen fibrils and disturbed mechanical properties of the tissues (Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H., Kadler, K. E., and Iozzo, R. V. (1997) J Cell Biol 136, 729-743; Svensson, L., Aszódi, A., Reinholt, F. P., Fäissler, R., Heinegård, D., and Oldberg, Å. (1999) J. Biol. Chem. 274, 9636-9647). A picture is emerging where proteins in the vicinity of the cell regulate the early stages of collagen fibre formation.
Perlecan exists as HS and CS substituted forms and it has been shown that these forms can be used to facilitate collagen fibril formation. To our surprise, the addition of free CS-E was effective in collagen fibril formation, but none of the other CS variants had any significant effect (e.g. CS-D or CS-6).
A number of publications describing the effect of chondroitin sulphate on wound healing and for treating arthrosis exist (U.S. Pat. No. 5,929,050, JP10120577 and RU2216332). The present invention differs from these significantly as the use of CS-E or active fragments thereof stimulates the formation of collagen based extracellular matrix (ECM) and thus acting as fibrillogenesis agonists or, by modification of CS-E or active fragments thereof, as fibrosis antagonists.